Necrotizing enterocolitis (NEC) is the most common acute gastrointestinal emergency in premature infants with a mortality of 20-35%. Although aberrant activation of the Toll-Like Receptor (TLR) family of immune receptors by the gut microbiota is a central event in NEC pathogenesis, the mechanisms underlying TLR pathway activation selectively in infants who develop NEC remain unknown. Our laboratory used an immunogenetics approach to discover Single Immunoglobulin Interleukin-1-Related Receptor (SIGIRR), a negative regulator of TLR signaling, as a potential locus for NEC susceptibility. Pilot studies from our lab demonstrate that SIGIRR variants identified in human NEC result in a loss of inhibition of TLR and interleukin-1 receptor signaling in intestinal epithelial cells. Interestingly, unweaned mice with a knock-in of the mutation we identified in human NEC exhibit spontaneous activation of pro-inflammatory TLR signaling and intestinal dysbiosis. Based on these preliminary studies, we hypothesize that SIGIRR is a pathogenic locus for NEC susceptibility in premature infants, and deleterious SIGIRR mutations program deviant TLR4-microbiota interactions underlying NEC pathogenesis. This hypothesis will be tested by: 1) determining whether SIGIRR variants increase NEC risk in premature infants, and examining the mechanisms by which pathogenic variants disrupt molecular function, and 2) investigating the mechanisms by which SIGIRR mutations program TLR hyperresponsiveness and dysbiosis in NEC using novel mice with a knock-in of the SIGIRR mutation identified in human NEC. In Aim 1, the SIGIRR genetic locus will be sequenced in premature infants with and without NEC to determine whether rare or novel SIGIRR variants contribute to NEC susceptibility or severity. A sequence-to-structure-to-function analysis of SIGIRR combining bioinformatics analysis of SIGIRR protein structure with in vitro functional assays of genetic variants will reveal the molecular basis of altered SIGIRR function with NEC variants. For Aim 2, we created SIGIRRTIR-/- mice with a knock-in of the p.Y168X mutation identified in human NEC to elucidate the mechanisms underlying deviant microbiota-TLR4 interactions in NEC. We will characterize the molecular players and microbiota signatures that direct native TLR hyperresponsiveness and NEC vulnerability. We will also test the efficacy of probiotics and TLR4-antagonists to mitigate NEC in SIGIRRTIR-/- mice. The rationale here is to investigate the paradigm that genetically-mediated defects in SIGIRR function program deviant microbiota-TLR interactions in NEC. We have taken a translational bedside-bench approach to delineate the mechanistic role of SIGIRR mutations in NEC pathogenesis. Results of this study will significantly advance knowledge in the areas of SIGIRR biology, genetic basis of NEC susceptibility, and regulation of neonatal gut microbiome by SIGIRR. These scientific gains will positively impact the existing status quo relating to the paucity of personalized screening tools for NEC prediction, and new treatment strategies for NEC prevention in premature infants.